The long range goal of the proposed work is to define the molecular components and mechanisms mediating Vibrio cholerae colonization and virulence protein secretion to the point where there is sufficient knowledge to intelligently incorporate this information into improved cholera vaccine strategies and antimicrobial therapies designed to inhibit these events. Most of the proposal involves analysis of the molecular mechanisms by which toxin coregulated pilus (TCP) is formed and mediates intestinal colonization. Some steps in the process by which TCP and other type 4 pili are built are linked and/or related to the process of toxin and other virulence determinant secretion by type II secretion systems. Thus further understanding of the mechanisms of type 4 pilus biogenesis should lead to the characterization of potential antimicrobial targets involved in multiple virulence pathways. We will examine the aspects of pilus biogenesis in detail. These experiments will be facilitated by our currently available collections of tcp genetic constructs and immunoreagents. Regarding the mechanism of TCP function, we will utilize the technique of field emission scanning electron microscopy in combination with specifically engineered tcpA missense mutations to address the biophysical mechanisms that lead to pilus supertwist formation, promoting the bacterial associations that represent the basis of microcolony formation in the intestine. Further characterization of TcpF and its role in colonization will be undertaken. TcpF has become even more interesting with the identification by other labs of genes in two other pilus operons that are located in the same position as tcpF and that encode secreted proteins that have no homology amongst themselves. Lastly we will build upon our identification of a new V. cholerae colonization factor that links the epithelial steps of intestinal colonization with environmental persistence and quorum sensing to understand the mechanism by which the new factor, GbpA, functions and is modulated by the quorum sensing system. Additional steps in the epithelial interaction with V. cholerae will be defined. Taken together, the results of the proposed studies will provide detailed information regarding the mechanisms of V. cholerae colonization from the levels of defining the components, understanding how they are elaborated to the cell surface and beyond, and even addressing the biophysical nature of pilus interactions that are essential for microcolony formation and disease.